Method for producing transformed plant having increased glutamic acid content

ABSTRACT

An object of the present invention is to provide a method for producing a transformed plant whose free glutamic acid content is increased. Moreover, it is a further object of the present invention to provide a transformed plant, whose free glutamic acid content is increased, progeny plants thereof and seeds thereof.  
     The method of the present invention comprises the steps of transforming a plant with a nucleic acid construct capable of controlling the expression of OGDH gene, and then selecting or identifying the transformed plants based on the presence of the expression of a marker gene present on the nucleic acid construct to thus screen the plant having an increased free glutamic acid content.

BACKGROUND OF THE INVENTION

[0001] The present invention relates to a method for the production of atransformed plant whose free glutamic acid content is increased.

[0002] Glutamic acid as an α-amino acid is in general extensivelypresent in proteins and has been known as a component governing thetaste and flavor of tomato and components governing the taste and flavorof fermented foods made of, for instance, soy beans. It has been knownthat glutamic acid is synthesized at the initial stage of the nitrogenassimilation in the higher plant, thereafter glutamine or asparagineoriginated from glutamic acid are transported to every tissues of theplant body through the sieve tube thereof and then they are used in thesynthesis of other amino acids and proteins in the tissues. As has beendiscussed above, glutamic acid is an amino group donor and ismetabolized in a variety of biosynthesis pathways. Accordingly, it isnot easy to increase the free glutamic acid content in the plant bodyand therefore, there have conventionally been known only a small numberof examples in which the free glutamic acid content in, for instance,roots of tobacco or corn plants is increased by the incorporation of agene coding for glutamate dehydrogenase into the same.

[0003] Increasing the content of 2-oxoglutaric acid has been consideredas a means for increasing the free glutamic acid content in plant'sbodies. This is because, the glutamine synthetase-glutamic acid synthasepathway requires 2-oxoglutaric acid as a carbon skeleton. In particular,as an effective means for increasing the free glutamic acid content inmicroorganisms, there has been proposed a method for inhibiting anenzyme or 2-oxoglutarate dehydrogenase (OGDH), which oxidizes2-oxoglutaric acid in the TCA cycle to thus produce succinylCoA(Japanese Un-Examined Patent Publication No. Hei 7-203980). Accordingto this report, in mutants where OGDH activity is defective or reduced,the pathway from 2-oxoglutaric acid to succinyl CoA in the TCA cycle isinhibited, and therefore, the progress of the biosynthesis pathway from2-oxoglutaric acid to succinyl CoA is improved and the mutant isimproved in the ability of producing glutamic acid.

[0004] However, it has been known that the production of glutamic acidfrom 2-oxoglutaric acid in the plant is taken place in the chloroplastand that the glutamic acid-producing site in the plant is considerablydifferent from that observed for the foregoing microorganisms. As the2-oxoglutaric acid supply pathway in the plant, there has been known apathway in the cytoplasm wherein 2-oxoglutaric acid is likewisesynthesized by the action of isocitrate dehydrogenase (ICDH) from citricacid, through isocitric acid, in addition to the TCA cycle in themitochondrion or citric acid-isocitric acid-2-oxoglutaric acid pathwayin the TCA cycle. It has been reported that the activity of cytoplasmisocitrate dehydrogenase reaches 95% of the total activity of ICDH intobacco-chlorenchyma and that the citric acid-isocitricacid-2-oxoglutaric acid pathway mainly contributes to the glutamic acidproduction in the plant's chloroplast. On the other hand, there has beenknown such a report that, in the tobacco whose ICDH of the cytoplasm isinhibited by an antisense RNA, the concentrations of 2-oxoglutaric acidand free glutamic acid therein do not undergo any change (Kruse, A. etal., Planta, 1998, 205:82-91). However, the pathway from 2-oxoglutaricacid to succinyl CoA, in connection with the biosynthesis pathway ofglutamic acid has not yet been regarded as important and thus the OGDHgene involved in this pathway has not positively been geneticallyengineered. The OGDH gene has not been subjected to any gene engineeringas described above, while although the genome structures of at least twogenes presumed to be OGDH genes of plants have been known for OGDH E1subunit, any correct cDNA sequence for the one gene has not yet beenknown and in any case, there have not yet been investigated functions ofthe proteins encoded by the genes and roles thereof in the glutamic acidproduction.

SUMMARY OF THE INVENTION

[0005] It is an object of the present invention to provide a method forproducing a transformed plant whose free glutamic acid content isincreased. Moreover, it is a further object of the present invention toprovide a transformed plant whose free glutamic acid content isincreased, progeny plants thereof and seeds thereof.

[0006] The inventors of this invention have paid attention to2-oxoglutaric acid produced in a mitochondrion and the role of2-oxoglutarate dehydrogenase (OGDH), have found that the free glutamicacid content in plants can be increased by the inhibition of thisactivity, in particular, the inhibition of the expression of OGDH E1subunits and thus have completed the present invention.

[0007] Accordingly, the present invention relates to a method forproducing a transformed plant whose free glutamic acid content isincreased as compared with a naturally occurring plant of the samespecies cultivated under the same conditions, which comprises the stepof inhibiting the expression of a gene coding for 2-oxoglutaratedehydrogenase.

[0008] More specifically, the present invention relates to a method forproducing a transformed plant whose free glutamic acid content isincreased as compared with a naturally occurring plant of the samespecies cultivated under the same conditions, which comprises the stepsof transforming a plant with a nucleic acid construct for controllingthe expression of OGDH, and then screening the transformed plant basedon the expression of a marker gene present on the nucleic acid constructas an indication to thus select or identify the transformed plant havingan increased free glutamic acid content.

[0009] In particular, the present invention relates to a methodcharacterized in that the foregoing nucleic acid construct is onecomprising an antisense RNA against the OGDH E1 subunit gene and aregulator sequence, which can express the antisense RNA.

[0010] Moreover, the present invention also relates to progeny plantswhose free glutamic acid content is increased and which are originatedfrom the transformed plant whose free glutamic acid content is increasedand which is produced by the foregoing method as well as seeds of theprogeny plants wherein a nucleic acid construct for controlling theexpression of OGDH is operably incorporated into the genome thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011]FIG. 1 shows the structure of a plasmid pSA1AS1.

[0012]FIG. 2 shows the structure of a plasmid pBIKm-SA1AS1.

[0013]FIG. 3 shows the free amino acids contents in a transformed plantwhose ogd1 expression is inhibited.

[0014]FIG. 4 shows the overall amino acids contents of a transformedplant (OGD1 No. 3) whose ogd1 expression is inhibited.

[0015]FIG. 5 shows the 2-oxoglutalate content of a transformed plant(OGD1 No. 3) whose ogd1 expression is inhibited.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] An object of the present invention is to increase the freeglutamic acid content in plants. To this end, the present inventioninhibits the expression of a gene coding for 2-oxoglutaratedehydrogenase (OGDH), which converts 2-oxoglutaric acid into succinylCoA in the TCA cycle. Such inhibition can be effected by transforming aplant with a nucleic acid construct for inhibiting the expression ofOGDH such as that described in this specification. The resultingtransformed plant and the progeny plants thereof are screened on thebasis of, for instance, the expressed amount of mRNA corresponding toOGDH, the amount of OGDH protein, and the free glutamic acid content inthe plant body.

[0017] In short, the transformed plant whose free glutamic acid contentis increased according to the present invention can be produced by theprocedures given below:

[0018] a) Incorporating a nucleic acid construct for inhibiting theexpression of 2-oxoglutarate dehydrogenase (OGDH) into a plant cell or aplant body and selecting the resulting transformant;

[0019] b) Depending on the purposes, regenerating the transformant intoa plant body or harvesting seeds to thus obtain transformed plants;

[0020] c) Screening the transformed plants obtained in the step b) andfurther selecting the transformed plant whose free glutamic acid contentis increased;

[0021] d) If necessary, collecting the progeny plants or seeds of thetransformed plants obtained in the step c);

[0022] e) Evaluating the progeny plants or seeds obtained in the step d)for the free glutamic acid content to thus obtain progeny plants orseeds of the plants whose free glutamic acid content is increased.

[0023] In the present invention, the expression of the OGDH gene isinhibited. The expression of all of the genes coding for every subunitsconstituting OGDH may be inhibited, but the expression of only one kindof subunit may be inhibited. In general, it is sufficient to inhibit theexpression of only one gene coding for one subunit, but the expressionof a plurality of subunits may be inhibited depending on the plantspecies to be engineered and the degree of desired effects. Whenintensively inhibiting the expression, it is preferred to inhibit theexpression of subunits, which are not common to other enzymes or factorsplaying important roles in other metabolic systems or which interacttherewith to only a small extent, among the subunits constituting OGDH.The inhibition of the expression may be carried out at thetranscriptional level including the disruption of a gene or at thepost-transcriptional level, but it is preferred to control the degree ofthe inhibition. The inhibition of the expression can likewise be carriedout according to the expression of an antisense RNA or so-called genesilencing methods such as the expression of gene-specific doublestranded RNA (Chiou-FenCHuang et al., PNAS, 2000, 97:4985-4990) and theco-suppression through intensive expression of the sense sequence (ThePlant Journal, 1998, 16:651-659). In any case, the overall length or apart of the OGDH gene can be used. The term “OGDH gene” used in thisspecification means a generic name of genes coding for any of OGDHsubunits unless otherwise specified. Moreover, the term “OGDH mRNA”means a generic name of mRNA's corresponding to each subunit unlessotherwise specified.

[0024] For instance, the antisense sequence can be expressed bycombining an appropriate promoter with the full length or a part of theOGDH gene in the antisense direction or the direction opposed to theoriginal direction of transcription. The double stranded RNA can beformed by conversely arranging the full length or a part of the OGDHgenes embracing an appropriate spacer sequence therebetween andconnecting them to an appropriate promoter to thus make it express. Inthis case, the transcribed RNA forms the double stranded RNA in such amanner that the spacer sequence undergoes looping out. The objects ofthe present invention can be accomplished by incorporating, into a plantcell or a plant body, a nucleic acid construct, which allows such anantisense sequence or the double stranded RNA to express. In case wherethe co-suppression is employed, it is sufficient to make the full gengthor a part of the OGDH gene express under the control of a strongpromoter. In the method of the present invention, it is preferred to usethe inhibition of expression by the antisense RNA from the viewpoint of,for instance, the simplicity of operations.

[0025] In this connection, the term “antisense RNA” used in thisspecification is used for representing a RNA sequence including asequence complementary to a mRNA or a part thereof. Therefore, if theterm “antisense RNA” is used in connection with a specific gene, theterm includes RNA containing only a sequence complementary to the fulllegth sequence of mRNA naturally transcribed from the gene, RNAincluding only a sequence complementary to a part of the mRNA and RNA'sfurther including a sequence un-complementary to the mRNA in addition tothe foregoing sequences. Moreover, the “antisense RNA” may have aplurality of copies of a sequence complementary to mRNAs.

[0026] The nucleic acid construct usable in the present invention can beprepared using a method well-known to those skilled in the art.Regarding molecular biological means including the isolation of thenucleic acid construct and the method for determining the sequencethereof, one can refer to articles such as Sambrook et al., MolecularCloning-Laboratory manual, 2^(nd) edition, Cold Spring Harbor LaboratoryPress. In addition, the production of the nucleic acid construct used inthe present invention would often require the gene amplificationrepresented by the PCR method. Such methods are detailed in, forinstance, F. M.

[0027] Ausubel et al. (eds), Current Protocols in Molecular Biology,John Wiley & Sons, Inc. (1994).

[0028] In general, the nucleic acid construct used in the presentinvention may further contain, in addition to the OGDH gene or a partthereof, an appropriate promoter functional in plant cells such as anopaline synthase gene or 35S promoter of cauliflower mosaic virus, anappropriate terminator such as the terminator of nopaline synthase gene,other sequences required or effective for the expression, and a markergene used for screening the desired transformant, for instance,drug-resistant gene such as kanamycin-resistant gene, G418-resistantgene and hygromycin-resistant gene.

[0029] The promoter usable in such a construct may be a constitutivepromoter or may be organ-specific or growth stage-specific one and itmay be selected depending on the host used, the desired amount of theexpression and the organ or the growth stage in which the expression isparticularly intended. In a preferred embodiment of the presentinvention, there are used strong promoters, which undergo expressionun-specific to organs and growth stages and a CaMV35S promoter is, forinstance, used as such a promoter. Examples of organ-specific promotersusable herein are promoters of ribulose bisphosphate carboxylase(RuBisCo) gene and chloroplast a/b bonding protein gene. The mostpreferred embodiment of the present invention makes use of an OGDH genesequence connected to a strong constitutive promoter such as a CaMV35Spromoter in the antisense direction.

[0030] The method for introducing a gene usable in the present inventionis not restricted to any particular one and the methods known to thoseskilled in the art as ones for introducing a gene into plant cells orplant bodies can be selected depending on the host selected. Forinstance, a gene-introduction method, using Agrobacterium, is employedin an embodiment of the present invention. A binary vector is desirablyused in such a transformant line. In case where Agrobacterium is used,the sequence to be introduced is inserted between the left and rightT-DNA border sequences. The appropriate design and construction of sucha transforming vector based on the T-DNA has been well known to thoseskilled in the art. Moreover, the conditions for infecting a plant withAgrobacterium containing such a nucleic acid construct have also beenwell known to those skilled in the art. As to such techniques andconditions, one can refer to Cell Technology, a separate volume entitled“Experimental Protocol for Model Plants: Edition for Rice Plant andArabis thaliana” (1996).

[0031] In the present invention, other gene-introduction methods canlikewise be used. Examples of such gene-introduction methods usableherein are DNA-introduction methods, which make use of polyethyleneglycols and calcium; methods for transforming protoplasts according tothe electroporation; and transduction methods according to the particlegun.

[0032] Plant species, which are subjected to the foregoing geneengineering are not restricted to any specific one, but preferably usedherein are plant species whose transformation is easy and whose systemfor regenerating into the plant bodies has been established. Plantssuitably used in the present invention more preferably include, inaddition to those possessing the foregoing characteristic properties,plant species for which cultivation techniques for mass-production havebeen established from the viewpoint of the use of glutamic acidproduced. Specific examples of plants suitably used for carrying out thepresent invention include, in addition to all of the plants belonging tothe Cruciferae family, tomato, potato, corn, wheat, rice plants andsugar cane. Moreover, organs and cells, which are subjected to theforegoing gene engineering, are not restricted to any specific one andmay be selected depending on, for instance, the specific host selectedand the specific gene-introduction method selected. Specific examplesthereof are explants of organs, pollens, cultured cells, embryos andplant bodies, but the present invention is not restricted to thesespecific ones.

[0033] Then plant cells or the like engineered according to theforegoing procedures are screened for the transformants thereof. Thisselection can be effected on the basis of the expression of the markergene present on the nucleic acid construct used in the transformation.If the marker gene is, for instance, a drug resistant gene, theselection can be carried out by cultivating or growing the plant cellsor the like genetically engineered on a culture medium containing anantibiotic or a herbicide in an appropriate concentration.Alternatively, if the marker gene is, for instance, β-glucuronidase geneor luciferase gene, desired transformants can be selected by carryingout screening for the activity thereof. If the transformant thusidentified is a subject other than plant body such as a protoplast, acallus or an explant, it is regenerated into a plant body. Thisregeneration can be carried out using a method, known to those skilledin the art, and used for each particular host plant used. The plant bodythus obtained may be cultivated according to the conventional method orunder the same conditions used for the un-transformed plant body andthen subjected to a procedures for identifying the transformed plantcontaining the nucleic acid construct according to the present inventionsuch as those, which make use of a variety of molecular biologicaltechniques, in addition to the aforementioned selection on the basis ofthe marker gene. Such techniques usable in the present inventioninclude, for instance, Southern hybridization or PCR techniques for thedetection of the presence of a recombinant DNA insert and the structurethereof and Northern blotting and RT-PCR techniques for the detection ordetermination of an RNA transcript originated from the introducednucleic acid construct.

[0034] Then the resulting transformed plants are evaluated for theamount of OGDH or each OGDH subunit protein or the amount of OGDH mRNA.For instance, the amount of a protein can be evaluated by a method suchas Western blotting method and that of the mRNA can be evaluated by amethod such as Northern blotting or quantitative RT-PCR method.Moreover, the OGDH activity can be determined by, for instance, themethod of Miller et al. (Miller et al., Biochem. J., 1999, 343:327-334).All of these methods have been well known to those skilled in the artand kits for easily carrying out these methods can likewise becommercially available.

[0035] The transformed plant whose reduction in the amount of the OGDHprotein, that of the OGDH subunit protein, the OGDH activity or theamount of the OGDH mRNA has thus been confirmed is further inspected forthe free glutamic acid content. The free glutamic acid content can beexamined by, for instance, breaking the transformed plant or a partthereof into pieces, extracting the plant pieces to give an extract andthen treating the extract with an amino acid analyzer. Once atransformed plant whose free glutamic acid content is increased isidentified, the transformed plant is further inspected for whether thecharacters thereof are genetically stably maintained therein or not. Tothis end, it is sufficient to cultivate the plant body under the usualconditions, to harvest the seeds thereof and to analyze the charactersand separation thereof in the progeny thereof. The presence of theintroduced nucleic acid construct, the position thereof and theexpression thereof can be analyzed according to the same procedures usedfor analyzing the primary transformant.

[0036] In the transformed plant whose free glutamic acid content isincreased, the sequences originated from the nucleic acid constructintroduced and incorporated into the genome may be in the form of eitherheterozygote or homozygote. Eithere heterozygote or homozygote can beobtained by crossing or mating, depending on the needs. The sequencesoriginated from the nucleic acid construct incorporated into the genomewould be separated according to the Mendelian inheritance rule in theprogeny. For this reason, it is preferred to use the homozygous plant inorder to obtain progeny plants and seeds thereof from the viewpoint ofthe stability of the characters. The transformed plants thus obtainedcan be cultivated under the same cultivation conditions used for thenaturally occurring plant of the same species and glutamic acid can beextracted from the whole plants or each organ using the usual extractionconditions.

EXAMPLES Example 1

[0037] Preparation of OGDH E1 Subunit cDNA Originated from Arabidopsisthaliana

[0038] (1) Preparation of DNA, RNA

[0039] The seedlings of Arabidopsis thaliana (Columbia; Col-0) werecrushed into pieces together with glass beads (5 g/10 g tissues) in anextraction buffer (7.5 ml/10 g tissues; containing 0.2 M Tris HCI, pH8.0, 0.1 M EDTA, 1% Na N-laurylsarkosyl, 2 mg/ml proteinase K), followedby subjecting to the EtOH precipitation, separating DNA by a CsCIdensity gradient centrifugation and recovering the same. Separately, thewhole RNA was likewise prepared from the seedlings of Arabidopsisthaliana using a reagent ISOGEN for the preparation of RNA availablefrom Nippon Gene Co., Ltd. according to the attached protocol. ThenPolyA RNA was recovered from the resulting total RNA using Oligotex dT30Super available from Nippon Synthetic Rubber Co., Ltd.

[0040] (2) Preparation of Library

[0041] When searching for sequences homologous with SUCA amino acidsequence of Escherichia coli (E. coli ) using the published EST data ofArabidopsis thaliana, three sequences were obtained. These threesequences corresponded to 444-(T76109), 561-(H37127) and 682-(H36333) ofthe E. coli SUCA amino acid sequence. All of these EST sequences were onthe order of about 300 b in length and these three sequences did notoverlap one another.

[0042] Then to obtain a probe for an N-terminal proximal region, 5′-RACEwas carried out using polyA RNA prepared from the flower buds ofArabidopsis thaliana as a starting material and 5′ RACE System for RapidAmplification of cDNA Ends, Ver. 2.0 available from GIBCO BRL Company,according to the attached protocol. In this respect, there were used5′-GAAGGACAGAATGACGATGA-3′ (SEQ ID: 1) corresponding to the sequencederived from the 196 bases of EST H37127 as GSP1 (Gene Specific Primer),5′-GTGACGAGGGATGACTGCGT-3′ (SEQ ID: 2) corresponding to the sequencederived from the 110 bases of EST T76109 as GSP2 and5′-TCGTCTATCTCGTTATGCCC-3′ (SEQ ID:3) corresponding to the sequencederived from the 54 bases of EST T76109 as nested GSP. As a result, avariety of fragments were obtained and the longest one was found to be1.3 kb in length. The terminal of the fragment of 1.3 kb was bluntedbefore inserting the fragment into the Smal site of a plasmidpBluescript SK+. The resulting plasmid was digested with Sacl and BamHlto form a deletion plasmid and then the base sequence thereof wasdetermined. The resulting fragment was found to have a sequencehomologous with E. coli SucA.

[0043] (3) Isolation of OGDH Gene of Arabidopsis thaliana

[0044] A λ-phage cDNA library was prepared using polyA RNA prepared fromthe flower buds of Arabidopsis thaliana as a starting material andSUPERSCRIPT Lambda System for cDNA Synthesis and Lambda Cloning Kitavailable from GIBCO BRL Company according to the attached protocol.According to this kit, each cDNA fragment was inserted into theSall-Notl site of pZL1 contained in a vector λZ_(lP)Lox. Thus, a librarywas obtained, which comprised about 200,000 independent clones having avariety of lengths whose median was positioned at about 2 kb. Thislibrary was amplified and used in the following experiments.

[0045] A probe was prepared using Prime lt-II, [α-³²P]dCTP availablefrom Stratagene Company and a Hincll-Xbal digested fragment (a fragmentof about 500 bp) of the deletion plasmid prepared for sequencing the 5′RACE product obtained above, as a template. After carrying out plaquehybridization at 60° C. according to the method of Church et al. (ChurchG. M. et al., P.N.A.S. USA, 1984, 74:5350), the hybridization productwas washed with 0.2× SSC 0.1% SDS at 60° C. (Hybridization was likewisecarried out in the Northern and Southern analysis under the similarconditions, unless otherwise specified).

[0046] As a result, two clones were obtained, which comprised cDNA'shaving different sequences and a length of about 3.5 kb. The sequencesof cDNA's included in these clones were determined and it was found thatboth of them comprised sequences homologous with E. coli SucA. Inaddition, putative amino acid sequences were compared with OGDH E1subunits derived from organisms of other species and the upstreamsequences were analyzed. As a result, it was found that a stop codon ispresent in the same upstream reading frame and that it has such atypical sequence that in the proximity to the putative initiation codon,+4-position is G and −3-position is a purine. Accordingly, it wasrecognized that the cDNA contained in these clones encompass the fulllength of the translational region of the OGDH gene. From these results,it was suggested that the genes contained in these two clones are thetwo genes encoding for OGDH E1 subunits of Arabidopsis thaliana. Theputative amino acid sequences had a homology with OGDH's from otherspecies, of about 40%. These two genes will hereafter be referred to as“OGD1 gene” or “ogd1” and “OGD2 gene” or “ogd2”, respectively. Theplasmid in which each cDNA was inserted into the Sall-Notl region wasspliced from each clone corresponding to odg1 or odg2 using adeletion/recyclized system (the aforementioned kit available from GIBCOBR Company), which made use of the Cre-loxP recombination to thus give aplasmid pAtOGD1 or pAtOGD2. The base sequences of ogd1 and ogd2 cDNA'sincluded in these plasmids are shown in SEQ ID: 4 and SEQ ID:6, whilethe amino acid sequences presumed on the basis of these cDNA's arelikewise shown in SEQ ID: 5 and SEQ ID:7, respectively.

Example 2

[0047] Inhibition of Expression of OGDH Gene in Arabidopsis thaliana

[0048] (1) Construction of Plasmid for Antisense Sequence Expression

[0049] In this Example, pBIKm and pBINHYGTOR (Hofgen et al., P.N.A.S.,91:1726-1730) were used as binary vectors carrying a CaMV35S promoter.In this respect, pBIKm is one in which the uidA site of pBI121 issubstituted for the Smal-Sacl site of pUC18 polylinker. The plasmid forexpressing an antisense RNA corresponding to the whole length of ogd1cDNA was constructed according to the following procedures. AnEcoRV-Sall digestion fragment of the PCR products corresponding to theC-terminal portion of OGD1 obtained using pAtOGD1 discribed in Example 1as a template was introduced into the Sall site of pBluescriptSK+together with the Sall-EcoRV digested fragment (including the region onthe N-terminal region of OGD1) of the plasmid pAtODG1, while using aprimer: 5′-CGGGTCGACAGCAATAACAAAACTGTATA-3′ (SEQ ID: 9; the portion ofthe ogd1 sequence corresponds to the 3379^(th) to 3360^(th) nucleotidesin SEQ ID: 4) to which a primer: 5′-ATCTCATTCAGCGTGAGCTC-3′ (SEQ ID: 8;the portion corresponding to the 2900^(th) to 2919^(th) nucleotides inSEQ ID: 4) and the Sall site were added and the Sall fragment of theresulting plasmid was inserted into the plasmid pBINHYGTOR to give aplasmid pSA1AS1 (FIG. 1). After blunting the fragment, it was insertedinto the Smal site of pBIKm to give a plasmid pBIKm-Sa1AS1 (FIG. 2).These plasmids comprise the complete fragment corresponding to 1^(st) to3379th nulcleotides of SEQ ID: 4 and have a fragment in which the Salllinker is added to the 5′- and 3′-terminals.

[0050] After cloning all of the foregoing PCR products, the nucleotidesequences thereof were confirmed prior to the final plasmidconstruction.

[0051] (2) Introduction of Antisense Plasmid-Expressing Plasmid intoPlants

[0052] Two kinds of the foregoing plasmids were introduced intoAgrobacterium C58C1 Rif according to the triparental mating techniqueusing E. coli and helper E. coli strain HB101/pRK2013 carrying theplasmids pSA1AS1 and pBIKm-SA1AS1. A plant was then infected by theinfiltration under reduced pressure technique using the resultingAgrobacterium C58C1Rif carrying the plasmid pSA1AS1 or pBIKm-Sa1AS1according to the procedures disclosed in Cell Technology, a separatevolume entitled “Experimental Protocol for Model Plants: Edition forRice Plant and Arabis thaliana”, published by SHUJUNSHA Publishing Press(1996). In case of the infiltration under reduced pressure technique,the seeds harvested from the infiltrated plant correspond to the T1plants and therefore, the primary screening thereof was carried out byseeding these sterilized seeds on a GM agar medium (1×MS, 1×B5 vitamin,10 g/l sucrose, 0.5 g/l MES-KOH (pH 7.5), 0.8% agar) containing 20 mg/lof hygromycin B (or 50 mg/l of Kanamycin sulfate). T2 Seeds wereharvested from the-plants grown on the culture medium. The T1 plant is aheterozygote and therefore, the T2 plants grown from the T2 seeds shouldundergo separation in accordance with the Mendel's law. In fact, this isalso true in the experiments in this Example. Thus, a part of the seedsoriginated from the T2 plants were seeded to examine the resistance tohygromycin of the T3 plants and the line in which all of the T3 plantswere hygromycin-resistant was maintained as a homozygous line withrespect to the introduced gene.

[0053] Then genome Southern analysis was carried out using 8 lines (6homogeneous lines and 2 heterogeneous lines) of the T3 plants derivedfrom each T1line of plants, which were expected to have a sequenceconstructed so as to express an RNA having the ogd1 antisense sequence.The probe used herein was PCR products obtained using primers5′-CGGGTACCCAAGTGTAGAACGACGATTG-3′ (SEQ ID: 10) and5′-CGTCTAGATGGTCGGTTCTCAGACATGA-3′ (SEQ ID: 11) and using pOGD1 as atemplate. After digesting 200 ng of genome DNA with Hind III, Southernblotting analysis was performed using the foregoing fragment as a probe.In this respect, the hybridization was effected according to the methodof Church et al. (Church, G. M. et al., P.N.A.S. USA, 1984, 74:5350). Asa result, it was confirmed that the sequence used for expressing theantisense sequence was incorporated into the genome.

[0054] (3) Evaluation of Amounts of mRNA and Protein of OGDH E1 Subunit

[0055] The amount of mRNA present in ogd1 as one of genes coding forOGDH E1 subunits observed for the transformed plant was compared withthat observed for non-transformed plant. Then Northern blotting analysiswas effected using 10 μg of the whole RNA prepared from the rosetteleaves of Arabidopsis thaliana. The antisense probe or the probe fordetecting ogd1 mRNA was prepared by digesting a plasmid constructed byintroducing the same PCR product used in the Southern blotting analysisin Section (2) into the plasmid pBluescriptSK+ and using T3 polymerasewith In Vitro Transcription Kit available from Stratagene Company. Inthis connection, the hybridization was carried out using the bufferhaving a composition specified in the explanatory note attached to InVitro Transcription Kit available from Stratagene Company under theconditions described therein.

[0056] As a result, in 3 lines out of 6 lines, which were proved to behomozygotes based on the separation pattern of the hygromycin resistantplant body, there was observed a considerable signal reduction in theband of about 3.5 kb and this indicates that the amount of OGD1 mRNA issubstantially reduced.

[0057] Then these three lines in which it was proved, in the Northernblotting analysis, that OGD1 mRNA content was reduced were inspected forthe amount of ogd1 translated products or the amount of OGD1 proteins.

[0058] For the purpose of effecting Western blotting analysis, a rabbitanti-serum against a partial peptide of OGD1 expressed in E. coli hostwas prepared. We entrusted Takara Shuzo Co., Ltd. with the preparationthereof. In this respect, an antigen used herein was a peptide obtainedby adding Met-Ser-Phe- to the N-terminal of the OGD1 partial peptidecorresponding to the 38^(th) to 512^(th) residues of the amino acidsequence described in SEQ ID:5. The fragment: Met-Ser-Phe- is not asequence derived from OGD1, but a sequence artificially added theretoduring the construction of a partial peptide-expressing construct in E.coli.

[0059] The rosette leaf extracts from the foregoing 6 lines ofArabidopsis thaliana were subjected to Western blotting analysis usingthe resulting anti-serum. The proteins were transferred to a PVDFmembrane by carrying out SDS-polyacrylamide gel electrophoresisaccording to the method of Laemmli et al. The proteins were transferredusing the buffer of Towbin (Towbin, H. et al., P.N.A.S., 1979, 76:4350)in a Sartoblotil device available from Sartorius Company. The proteinson the membrane were detected using the foregoing anti-serum and ImmuneBlotting Kit available from Bio-Rad Company according to the methodrecommended by the manufacturer.

[0060] As a result, there was observed a decrease of the OGD1 content inthe lines whose mRNA content was reduced in the Northern analysis, butthere was not observed any reduction of the OGD1 content in the lines,which did not show any reduction of the mRNA content. More specifically,it was found that the reduction of the mRNA content accompanied by theantisense RNA expression (as determined in the Northern blottinganalysis) was strongly correlated with the reduction of the OGD1 proteincontent.

Example 3

[0061] Evaluation of Free Glutamic Acid Content, Overall Amino AcidContent and 2-Oxoglutaric Acid Content in Plant Whose Expression of OGDHE1 Subunit is inhibited

[0062] The transformed plant lines, which showed a decrease of the OGD1mRNA content were grown over 4 weeks in an air-conditioned greenhouse(photoperiod: 14 hours; temperature: 23° C.). The leaves of eachtransformed plant line were milled using a mortar and a pestle in liquidnitrogen, followed by extraction thereof with 80% ethanol at 70° C.,concentration of the extract from which proteins were removed throughultrafiltration, dilution of the concentrate with a 0.01 N hydrochloricacid solution and determination of the amount of free amino acids-usingan amino acid analyzer L8800 available from Hitachi, Ltd.

[0063] The extraction of keto acid and the derivatization with2,4-dinitrophenyl hydrazine were carried out according to the proceduresdisclosed in the literature (Slater et al., Nature Biotech.,17:1011-1016) and then free amino acids and 2-oxoglutaric acid wereseparated from one another and quantitatively analyzed using ODS-80TMavailable from Tosoh Corporation and 60% ethanol and 1.5% acetic acidsolutions as solvents.

[0064] Typical results of these determinations are summarized in thefollowing Tables 1 to 3 and plotted on FIGS. 3 to 5. TABLE 1 Free AminoAcid Composition (%) of Transformed Strain ogd1 Antisense RNA-IntroducedPlant Control Plant Line (OGD1 No.3) Asp 19.55 14.21 Thr 6.08 4.80 Ser13.07 11.57 Asn 1.44 1.91 Glu 37.16 47.68 Gly 1.07 1.13 Ala 5.31 4.48Val 3.51 3.56 Ile 0.72 0.63 Leu 1.15 1.03 Tyr 0.43 0.38 Phe 1.07 1.01GABA 0.80 0.80 Gln 6.33 4.63 Lys 0.69 0.65 His 1.07 0.91 Arg 0.56 0.63

[0065] TABLE 2 Overall Amino Acid Content of Transformed Strain(nmol/gFW) ogd1 Antisense RNA-Introduced Plant Control Plant Line (OGD1No.3) 2800 3000

[0066] TABLE 3 2-Oxoglutaric Acid Content of Transformed Strain(nmol/gFW) ogd1 Antisense RNA-Introduced Plant Control Plant Line (OGD1No.3) 84 68

[0067] The foregoing results indicate that the free glutamic acidcontent of a plant body is significantly increased in the line whoseexpression of ogd1 is inhibited by the action of ogd1 antisense RNA.

[0068] The present invention permits the production of a transformedplant whose free glutamic acid content is increased. The presentinvention can provide a transformed plant whose free glutamic acidcontent is increased by at least 20%.

[0069] It is also understood that the examples and embodiments describedherein are only for illustrative purpose, and that various modificationswill be suggested to those skilled in the art without departing from thespirit and the scope of the invention as hereinafter claimed.

1 11 1 20 DNA Artificial Sequence Synthetic DNA 1 gaaggacaga atgacgatga20 2 20 DNA Artificial Sequence Synthetic DNA 2 gtgacgaggg atgactgcgt 203 20 DNA Artificial Sequence Synthetic DNA 3 tcgtctatct cgttatgccc 20 43394 DNA Arabidopsis thaliana CDS (109)..(3159) 4 tttctcttct tcgcctcctcctcctccaag tgtagaacga cgattgttga atgcgttatt 60 gtaattgtta agaattgaagttaaagtgtt gtttttgaat ctggtgaa atg gtg tgg 117 Met Val Trp 1 ttt cgt gctggt tcc agt gtt aca aag cta gct gtt aga agg att ttg 165 Phe Arg Ala GlySer Ser Val Thr Lys Leu Ala Val Arg Arg Ile Leu 5 10 15 aat cag ggt gcttcg tat gcg acg agg aca cgg tct att ccg tct caa 213 Asn Gln Gly Ala SerTyr Ala Thr Arg Thr Arg Ser Ile Pro Ser Gln 20 25 30 35 act cgt tcc tttcac tcg act ata tgc aga cca aag gct cag agt gct 261 Thr Arg Ser Phe HisSer Thr Ile Cys Arg Pro Lys Ala Gln Ser Ala 40 45 50 cca gtt cct aga gctgtt cct ctt tct aag cta act gat agt ttc tta 309 Pro Val Pro Arg Ala ValPro Leu Ser Lys Leu Thr Asp Ser Phe Leu 55 60 65 gat ggg acg agc agt gtctac ctt gag gag tta caa agg gct tgg gaa 357 Asp Gly Thr Ser Ser Val TyrLeu Glu Glu Leu Gln Arg Ala Trp Glu 70 75 80 gct gat cct aac agt gta gatgag tct tgg gat aat ttc ttt agg aac 405 Ala Asp Pro Asn Ser Val Asp GluSer Trp Asp Asn Phe Phe Arg Asn 85 90 95 ttt gtt ggt cag gct gcc acg tctcct ggc atc tct ggg cag aca att 453 Phe Val Gly Gln Ala Ala Thr Ser ProGly Ile Ser Gly Gln Thr Ile 100 105 110 115 cag gag agt atg agg ctg ttgtta ctt gtt agg gct tat cag gtg aat 501 Gln Glu Ser Met Arg Leu Leu LeuLeu Val Arg Ala Tyr Gln Val Asn 120 125 130 ggt cac atg aaa gcg aag ttggat ccg tta ggt ttg gaa cag cga gag 549 Gly His Met Lys Ala Lys Leu AspPro Leu Gly Leu Glu Gln Arg Glu 135 140 145 atc cct gag gat ctt gac ttggct ctt tat gga ttc act gag gct gac 597 Ile Pro Glu Asp Leu Asp Leu AlaLeu Tyr Gly Phe Thr Glu Ala Asp 150 155 160 ctt gac aga gag ttc ttc ttgggg gtg tgg cag atg tca gga ttc atg 645 Leu Asp Arg Glu Phe Phe Leu GlyVal Trp Gln Met Ser Gly Phe Met 165 170 175 tct gag aac cga cca gtg cagacc ctt cgt tcc ata ttg aca agg ctc 693 Ser Glu Asn Arg Pro Val Gln ThrLeu Arg Ser Ile Leu Thr Arg Leu 180 185 190 195 gaa cag gca tac tgt gggaat atc gga ttt gag tat atg cac att gca 741 Glu Gln Ala Tyr Cys Gly AsnIle Gly Phe Glu Tyr Met His Ile Ala 200 205 210 gat cga gat aaa tgt aactgg ttg aga gaa aag att gag aca cca act 789 Asp Arg Asp Lys Cys Asn TrpLeu Arg Glu Lys Ile Glu Thr Pro Thr 215 220 225 cct tgg cgg tac aac agggag cgc cgt gag gtg att ctc gat cgg ctt 837 Pro Trp Arg Tyr Asn Arg GluArg Arg Glu Val Ile Leu Asp Arg Leu 230 235 240 gca tgg agt act cag ttcgag aat ttc tta gct acc aag tgg aca aca 885 Ala Trp Ser Thr Gln Phe GluAsn Phe Leu Ala Thr Lys Trp Thr Thr 245 250 255 gcc aaa aga ttt gga cttgag gga gga gaa tca tta att cct gga atg 933 Ala Lys Arg Phe Gly Leu GluGly Gly Glu Ser Leu Ile Pro Gly Met 260 265 270 275 aag gag atg ttt gacaga gca gca gat ctt gga gta gag agt att gtt 981 Lys Glu Met Phe Asp ArgAla Ala Asp Leu Gly Val Glu Ser Ile Val 280 285 290 att gga atg tct cacaga gga aga ttg aat gtt ctg ggt aat gtt gtt 1029 Ile Gly Met Ser His ArgGly Arg Leu Asn Val Leu Gly Asn Val Val 295 300 305 cgg aag cca ctc cgtcag ata ttt agt gag ttc agt ggt ggt att agg 1077 Arg Lys Pro Leu Arg GlnIle Phe Ser Glu Phe Ser Gly Gly Ile Arg 310 315 320 cct gta gat gaa gttggc tac act gga act ggt gat gtc aaa tat cac 1125 Pro Val Asp Glu Val GlyTyr Thr Gly Thr Gly Asp Val Lys Tyr His 325 330 335 ttg gga acc tct tatgat cga cct aca aga ggt ggg aag aaa atc cat 1173 Leu Gly Thr Ser Tyr AspArg Pro Thr Arg Gly Gly Lys Lys Ile His 340 345 350 355 ctc tct ttg gttgct aat cca agt cac ttg gaa gct gca gat tct gtt 1221 Leu Ser Leu Val AlaAsn Pro Ser His Leu Glu Ala Ala Asp Ser Val 360 365 370 gtt gtt ggc aaaacc aga gca aaa cag tac tac tcc aat gat tta gac 1269 Val Val Gly Lys ThrArg Ala Lys Gln Tyr Tyr Ser Asn Asp Leu Asp 375 380 385 agg acc aaa aattta ggt att ttg att cac gga gat ggt agt ttt gct 1317 Arg Thr Lys Asn LeuGly Ile Leu Ile His Gly Asp Gly Ser Phe Ala 390 395 400 gga caa ggg gtagtc tat gaa act ctc cat ctt agt gct ctt cca aac 1365 Gly Gln Gly Val ValTyr Glu Thr Leu His Leu Ser Ala Leu Pro Asn 405 410 415 tac acc acc ggagga acc ata cat att gtg gtg aac aac caa gtg gct 1413 Tyr Thr Thr Gly GlyThr Ile His Ile Val Val Asn Asn Gln Val Ala 420 425 430 435 ttc acg acagat cca agg gcg ggg aga tct tcc cag tat tgt act gat 1461 Phe Thr Thr AspPro Arg Ala Gly Arg Ser Ser Gln Tyr Cys Thr Asp 440 445 450 gtt gca aaggct ttg agt gct ccc atc ttt cat gtt aat ggg gat gat 1509 Val Ala Lys AlaLeu Ser Ala Pro Ile Phe His Val Asn Gly Asp Asp 455 460 465 gtt gag gctgtt gtt cat gcc tgc gag ctt gct gct gag tgg cgt cag 1557 Val Glu Ala ValVal His Ala Cys Glu Leu Ala Ala Glu Trp Arg Gln 470 475 480 act ttt cattct gat gtt gtc gtt gat ttg gtt tgc tac cgt agg ttc 1605 Thr Phe His SerAsp Val Val Val Asp Leu Val Cys Tyr Arg Arg Phe 485 490 495 ggg cat aatgag ata gat gaa cca tct ttc act cag cca aaa atg tac 1653 Gly His Asn GluIle Asp Glu Pro Ser Phe Thr Gln Pro Lys Met Tyr 500 505 510 515 aag gttatc aaa aat cat cct tca acc ctt cag atc tac cac aaa aag 1701 Lys Val IleLys Asn His Pro Ser Thr Leu Gln Ile Tyr His Lys Lys 520 525 530 ctc ttggaa tgc ggt gaa gta tca caa cag gat att gac cgg ata cag 1749 Leu Leu GluCys Gly Glu Val Ser Gln Gln Asp Ile Asp Arg Ile Gln 535 540 545 gaa aaggtt aac acc atc ctc aat gaa gaa ttt gtc gct agt aag gac 1797 Glu Lys ValAsn Thr Ile Leu Asn Glu Glu Phe Val Ala Ser Lys Asp 550 555 560 tat ctccct aag aaa cga gat tgg ctt tca acc aat tgg gct gga ttt 1845 Tyr Leu ProLys Lys Arg Asp Trp Leu Ser Thr Asn Trp Ala Gly Phe 565 570 575 aag tctcct gag cag atc tca cgt gtt aga aac act ggc gtc aaa cca 1893 Lys Ser ProGlu Gln Ile Ser Arg Val Arg Asn Thr Gly Val Lys Pro 580 585 590 595 gagata ctg aag act gtt ggc aag gca att tca tct ctt cca gaa aac 1941 Glu IleLeu Lys Thr Val Gly Lys Ala Ile Ser Ser Leu Pro Glu Asn 600 605 610 ttcaag cca cac agg gca gtg aag aaa gtt tat gaa caa cgt gcc caa 1989 Phe LysPro His Arg Ala Val Lys Lys Val Tyr Glu Gln Arg Ala Gln 615 620 625 atgatt gaa tca gga gag gga gtt gac tgg gcc ctt gca gaa gct ctt 2037 Met IleGlu Ser Gly Glu Gly Val Asp Trp Ala Leu Ala Glu Ala Leu 630 635 640 gctttt gct acc tta gtt gtg gaa ggc aat cat gtc cga ttg agt ggt 2085 Ala PheAla Thr Leu Val Val Glu Gly Asn His Val Arg Leu Ser Gly 645 650 655 caggat gtc gaa cga gga aca ttt agt cat cgt cat tct gtc ctt cat 2133 Gln AspVal Glu Arg Gly Thr Phe Ser His Arg His Ser Val Leu His 660 665 670 675gac cag gaa act gga gaa gag tat tgt cct cta gat cat ctc atc atg 2181 AspGln Glu Thr Gly Glu Glu Tyr Cys Pro Leu Asp His Leu Ile Met 680 685 690aat cag gat cct gag atg ttt act gtt agc aac agt tct ctt tca gaa 2229 AsnGln Asp Pro Glu Met Phe Thr Val Ser Asn Ser Ser Leu Ser Glu 695 700 705ttt ggt gtc ctt ggg ttc gaa ttg ggt tac tcc atg gaa agc ccg aac 2277 PheGly Val Leu Gly Phe Glu Leu Gly Tyr Ser Met Glu Ser Pro Asn 710 715 720tcg ttg gta cta tgg gaa gct cag ttt gga gac ttc gcc aat gga gct 2325 SerLeu Val Leu Trp Glu Ala Gln Phe Gly Asp Phe Ala Asn Gly Ala 725 730 735cag gtg ata ttt gat cag ttc atc agc agt gga gaa gcc aaa tgg ctg 2373 GlnVal Ile Phe Asp Gln Phe Ile Ser Ser Gly Glu Ala Lys Trp Leu 740 745 750755 cgt caa acc ggg ctt gtt atg cta ctt ccc cat ggt tat gat ggt cag 2421Arg Gln Thr Gly Leu Val Met Leu Leu Pro His Gly Tyr Asp Gly Gln 760 765770 gga cct gaa cat tca agt gcg agg ttg gaa cgt tac ctt cag atg agt 2469Gly Pro Glu His Ser Ser Ala Arg Leu Glu Arg Tyr Leu Gln Met Ser 775 780785 gat gat aat ccc tat gtc ata cca gac atg gaa cca aca atg cga aag 2517Asp Asp Asn Pro Tyr Val Ile Pro Asp Met Glu Pro Thr Met Arg Lys 790 795800 caa att caa gaa tgt aat tgg cag att gtc aat gcc aca act ccc gcc 2565Gln Ile Gln Glu Cys Asn Trp Gln Ile Val Asn Ala Thr Thr Pro Ala 805 810815 aac tat ttc cat gtt ctg cgg cga cag ata cac aga gac ttc cgt aag 2613Asn Tyr Phe His Val Leu Arg Arg Gln Ile His Arg Asp Phe Arg Lys 820 825830 835 cct ctg att gta atg gca cca aag aac ttg ctc cgt cac aag gac tgc2661 Pro Leu Ile Val Met Ala Pro Lys Asn Leu Leu Arg His Lys Asp Cys 840845 850 aaa tca aat ctc tca gag ttt gat gat gtc caa ggc cac cca ggt ttt2709 Lys Ser Asn Leu Ser Glu Phe Asp Asp Val Gln Gly His Pro Gly Phe 855860 865 gac aag caa gga act aga ttt aag cga tta atc aag gat cag aat gat2757 Asp Lys Gln Gly Thr Arg Phe Lys Arg Leu Ile Lys Asp Gln Asn Asp 870875 880 cac tct gat ctt gaa gaa ggc atc aga aga ttg gta ctt tgc tcc gga2805 His Ser Asp Leu Glu Glu Gly Ile Arg Arg Leu Val Leu Cys Ser Gly 885890 895 aag gtc tat tat gag ctt gat gat gaa cgg aag aag gtt ggc gca aca2853 Lys Val Tyr Tyr Glu Leu Asp Asp Glu Arg Lys Lys Val Gly Ala Thr 900905 910 915 gat gtt gct atc tgt aga gtt gaa cag ctt tgt cct ttc cca tatgat 2901 Asp Val Ala Ile Cys Arg Val Glu Gln Leu Cys Pro Phe Pro Tyr Asp920 925 930 ctc att cag cgt gag ctc aag aga tat cca aat gcg gag atc gtttgg 2949 Leu Ile Gln Arg Glu Leu Lys Arg Tyr Pro Asn Ala Glu Ile Val Trp935 940 945 tgc caa gaa gag gcg atg aac atg gga gca ttc agc tac ata tctcca 2997 Cys Gln Glu Glu Ala Met Asn Met Gly Ala Phe Ser Tyr Ile Ser Pro950 955 960 cgg cta tgg aca gca atg aga agc gta aac aga gga gat atg gaagac 3045 Arg Leu Trp Thr Ala Met Arg Ser Val Asn Arg Gly Asp Met Glu Asp965 970 975 att aag tat gtt ggt cgt ggt cct tct gct gca act gcc acg ggtttc 3093 Ile Lys Tyr Val Gly Arg Gly Pro Ser Ala Ala Thr Ala Thr Gly Phe980 985 990 995 tat act ttc cat gtc aaa gag caa gcc ggg ctt gtc cag aaagcc 3138 Tyr Thr Phe His Val Lys Glu Gln Ala Gly Leu Val Gln Lys Ala1000 1005 1010 atc gga aag gaa ccc atc aat taaaaactct tcctttttaaatgacttctc 3189 Ile Gly Lys Glu Pro Ile Asn 1015 agactgataa gagaaaaaggagaaaacttt gaaataagaa ctttgggtga tgcaaaagtg 3249 ctaaacaaca ctgtcaaaatccgctgtttc tttgatattt ttttggtccc attgattttg 3309 agcctggctc gtgttgccagtcaaaacatt aaaaaaaaat cttatattga tatacagttt 3369 tgttattgct aaaaaaaaaaaaaaa 3394 5 1017 PRT Arabidopsis thaliana 5 Met Val Trp Phe Arg Ala GlySer Ser Val Thr Lys Leu Ala Val Arg 1 5 10 15 Arg Ile Leu Asn Gln GlyAla Ser Tyr Ala Thr Arg Thr Arg Ser Ile 20 25 30 Pro Ser Gln Thr Arg SerPhe His Ser Thr Ile Cys Arg Pro Lys Ala 35 40 45 Gln Ser Ala Pro Val ProArg Ala Val Pro Leu Ser Lys Leu Thr Asp 50 55 60 Ser Phe Leu Asp Gly ThrSer Ser Val Tyr Leu Glu Glu Leu Gln Arg 65 70 75 80 Ala Trp Glu Ala AspPro Asn Ser Val Asp Glu Ser Trp Asp Asn Phe 85 90 95 Phe Arg Asn Phe ValGly Gln Ala Ala Thr Ser Pro Gly Ile Ser Gly 100 105 110 Gln Thr Ile GlnGlu Ser Met Arg Leu Leu Leu Leu Val Arg Ala Tyr 115 120 125 Gln Val AsnGly His Met Lys Ala Lys Leu Asp Pro Leu Gly Leu Glu 130 135 140 Gln ArgGlu Ile Pro Glu Asp Leu Asp Leu Ala Leu Tyr Gly Phe Thr 145 150 155 160Glu Ala Asp Leu Asp Arg Glu Phe Phe Leu Gly Val Trp Gln Met Ser 165 170175 Gly Phe Met Ser Glu Asn Arg Pro Val Gln Thr Leu Arg Ser Ile Leu 180185 190 Thr Arg Leu Glu Gln Ala Tyr Cys Gly Asn Ile Gly Phe Glu Tyr Met195 200 205 His Ile Ala Asp Arg Asp Lys Cys Asn Trp Leu Arg Glu Lys IleGlu 210 215 220 Thr Pro Thr Pro Trp Arg Tyr Asn Arg Glu Arg Arg Glu ValIle Leu 225 230 235 240 Asp Arg Leu Ala Trp Ser Thr Gln Phe Glu Asn PheLeu Ala Thr Lys 245 250 255 Trp Thr Thr Ala Lys Arg Phe Gly Leu Glu GlyGly Glu Ser Leu Ile 260 265 270 Pro Gly Met Lys Glu Met Phe Asp Arg AlaAla Asp Leu Gly Val Glu 275 280 285 Ser Ile Val Ile Gly Met Ser His ArgGly Arg Leu Asn Val Leu Gly 290 295 300 Asn Val Val Arg Lys Pro Leu ArgGln Ile Phe Ser Glu Phe Ser Gly 305 310 315 320 Gly Ile Arg Pro Val AspGlu Val Gly Tyr Thr Gly Thr Gly Asp Val 325 330 335 Lys Tyr His Leu GlyThr Ser Tyr Asp Arg Pro Thr Arg Gly Gly Lys 340 345 350 Lys Ile His LeuSer Leu Val Ala Asn Pro Ser His Leu Glu Ala Ala 355 360 365 Asp Ser ValVal Val Gly Lys Thr Arg Ala Lys Gln Tyr Tyr Ser Asn 370 375 380 Asp LeuAsp Arg Thr Lys Asn Leu Gly Ile Leu Ile His Gly Asp Gly 385 390 395 400Ser Phe Ala Gly Gln Gly Val Val Tyr Glu Thr Leu His Leu Ser Ala 405 410415 Leu Pro Asn Tyr Thr Thr Gly Gly Thr Ile His Ile Val Val Asn Asn 420425 430 Gln Val Ala Phe Thr Thr Asp Pro Arg Ala Gly Arg Ser Ser Gln Tyr435 440 445 Cys Thr Asp Val Ala Lys Ala Leu Ser Ala Pro Ile Phe His ValAsn 450 455 460 Gly Asp Asp Val Glu Ala Val Val His Ala Cys Glu Leu AlaAla Glu 465 470 475 480 Trp Arg Gln Thr Phe His Ser Asp Val Val Val AspLeu Val Cys Tyr 485 490 495 Arg Arg Phe Gly His Asn Glu Ile Asp Glu ProSer Phe Thr Gln Pro 500 505 510 Lys Met Tyr Lys Val Ile Lys Asn His ProSer Thr Leu Gln Ile Tyr 515 520 525 His Lys Lys Leu Leu Glu Cys Gly GluVal Ser Gln Gln Asp Ile Asp 530 535 540 Arg Ile Gln Glu Lys Val Asn ThrIle Leu Asn Glu Glu Phe Val Ala 545 550 555 560 Ser Lys Asp Tyr Leu ProLys Lys Arg Asp Trp Leu Ser Thr Asn Trp 565 570 575 Ala Gly Phe Lys SerPro Glu Gln Ile Ser Arg Val Arg Asn Thr Gly 580 585 590 Val Lys Pro GluIle Leu Lys Thr Val Gly Lys Ala Ile Ser Ser Leu 595 600 605 Pro Glu AsnPhe Lys Pro His Arg Ala Val Lys Lys Val Tyr Glu Gln 610 615 620 Arg AlaGln Met Ile Glu Ser Gly Glu Gly Val Asp Trp Ala Leu Ala 625 630 635 640Glu Ala Leu Ala Phe Ala Thr Leu Val Val Glu Gly Asn His Val Arg 645 650655 Leu Ser Gly Gln Asp Val Glu Arg Gly Thr Phe Ser His Arg His Ser 660665 670 Val Leu His Asp Gln Glu Thr Gly Glu Glu Tyr Cys Pro Leu Asp His675 680 685 Leu Ile Met Asn Gln Asp Pro Glu Met Phe Thr Val Ser Asn SerSer 690 695 700 Leu Ser Glu Phe Gly Val Leu Gly Phe Glu Leu Gly Tyr SerMet Glu 705 710 715 720 Ser Pro Asn Ser Leu Val Leu Trp Glu Ala Gln PheGly Asp Phe Ala 725 730 735 Asn Gly Ala Gln Val Ile Phe Asp Gln Phe IleSer Ser Gly Glu Ala 740 745 750 Lys Trp Leu Arg Gln Thr Gly Leu Val MetLeu Leu Pro His Gly Tyr 755 760 765 Asp Gly Gln Gly Pro Glu His Ser SerAla Arg Leu Glu Arg Tyr Leu 770 775 780 Gln Met Ser Asp Asp Asn Pro TyrVal Ile Pro Asp Met Glu Pro Thr 785 790 795 800 Met Arg Lys Gln Ile GlnGlu Cys Asn Trp Gln Ile Val Asn Ala Thr 805 810 815 Thr Pro Ala Asn TyrPhe His Val Leu Arg Arg Gln Ile His Arg Asp 820 825 830 Phe Arg Lys ProLeu Ile Val Met Ala Pro Lys Asn Leu Leu Arg His 835 840 845 Lys Asp CysLys Ser Asn Leu Ser Glu Phe Asp Asp Val Gln Gly His 850 855 860 Pro GlyPhe Asp Lys Gln Gly Thr Arg Phe Lys Arg Leu Ile Lys Asp 865 870 875 880Gln Asn Asp His Ser Asp Leu Glu Glu Gly Ile Arg Arg Leu Val Leu 885 890895 Cys Ser Gly Lys Val Tyr Tyr Glu Leu Asp Asp Glu Arg Lys Lys Val 900905 910 Gly Ala Thr Asp Val Ala Ile Cys Arg Val Glu Gln Leu Cys Pro Phe915 920 925 Pro Tyr Asp Leu Ile Gln Arg Glu Leu Lys Arg Tyr Pro Asn AlaGlu 930 935 940 Ile Val Trp Cys Gln Glu Glu Ala Met Asn Met Gly Ala PheSer Tyr 945 950 955 960 Ile Ser Pro Arg Leu Trp Thr Ala Met Arg Ser ValAsn Arg Gly Asp 965 970 975 Met Glu Asp Ile Lys Tyr Val Gly Arg Gly ProSer Ala Ala Thr Ala 980 985 990 Thr Gly Phe Tyr Thr Phe His Val Lys GluGln Ala Gly Leu Val Gln 995 1000 1005 Lys Ala Ile Gly Lys Glu Pro IleAsn 1010 1015 6 3412 DNA Arabidopsis thaliana CDS (78)..(3152) 6ttcagagatt aaacaatttg aaaaatcgga gactctggga ttgtatggtt cgttgttact 60gatagattac ttaagct atg gtt tgg ttt aga atc ggt tct tct gtg gca 110 MetVal Trp Phe Arg Ile Gly Ser Ser Val Ala 1 5 10 aag ctt gcc ata aga aggaca ctg tct cag tct cgt tgt ggt tca tat 158 Lys Leu Ala Ile Arg Arg ThrLeu Ser Gln Ser Arg Cys Gly Ser Tyr 15 20 25 gcc act aga aca agg gtt ttgcct tgt caa acc aga tgt ttt cac tct 206 Ala Thr Arg Thr Arg Val Leu ProCys Gln Thr Arg Cys Phe His Ser 30 35 40 aca ata ctc aaa tca aag gca gagtct gct gca cct gtt cca cgt cct 254 Thr Ile Leu Lys Ser Lys Ala Glu SerAla Ala Pro Val Pro Arg Pro 45 50 55 gtc cca ctt tct aag cta act gat agcttc tta gat gga aca agc agt 302 Val Pro Leu Ser Lys Leu Thr Asp Ser PheLeu Asp Gly Thr Ser Ser 60 65 70 75 gtg tat cta gag gag tta caa aga gcttgg gag gct gat ccc aac agt 350 Val Tyr Leu Glu Glu Leu Gln Arg Ala TrpGlu Ala Asp Pro Asn Ser 80 85 90 gtt gat gag tcg tgg gat aac ttt ttt aggaat ttt gtg ggt cag gct 398 Val Asp Glu Ser Trp Asp Asn Phe Phe Arg AsnPhe Val Gly Gln Ala 95 100 105 tct aca tcg cct ggt atc tcg ggg caa accatt caa gaa agc atg cgt 446 Ser Thr Ser Pro Gly Ile Ser Gly Gln Thr IleGln Glu Ser Met Arg 110 115 120 ttg ttg ttg cta gtt aga gct tac cag gttaat ggc cac atg aag gcc 494 Leu Leu Leu Leu Val Arg Ala Tyr Gln Val AsnGly His Met Lys Ala 125 130 135 aag ctt gat cct tta ggt cta gag aag agagag att cca gag gat ctc 542 Lys Leu Asp Pro Leu Gly Leu Glu Lys Arg GluIle Pro Glu Asp Leu 140 145 150 155 acg cca ggt ctt tat ggg ttt act gaggct gat ctt gat cgg gaa ttc 590 Thr Pro Gly Leu Tyr Gly Phe Thr Glu AlaAsp Leu Asp Arg Glu Phe 160 165 170 ttt ctg ggt gta tgg agg atg tcg ggtttt ctc tct gag aac cgc ccg 638 Phe Leu Gly Val Trp Arg Met Ser Gly PheLeu Ser Glu Asn Arg Pro 175 180 185 gtt caa aca ctg agg tcg ata ctg tcgagg ctt gag caa gct tac tgt 686 Val Gln Thr Leu Arg Ser Ile Leu Ser ArgLeu Glu Gln Ala Tyr Cys 190 195 200 ggg act ata ggg tat gag tac atg cacatt gct gat agg gat aaa tgt 734 Gly Thr Ile Gly Tyr Glu Tyr Met His IleAla Asp Arg Asp Lys Cys 205 210 215 aac tgg ttg aga gac aag atc gag acccca act cct cga cag tac aat 782 Asn Trp Leu Arg Asp Lys Ile Glu Thr ProThr Pro Arg Gln Tyr Asn 220 225 230 235 agt gag cgt cgg atg gtt att tatgat agg ctt acc tgg agc aca cag 830 Ser Glu Arg Arg Met Val Ile Tyr AspArg Leu Thr Trp Ser Thr Gln 240 245 250 ttt gag aat ttc ttg gct act aagtgg acc acg gct aaa agg ttt gga 878 Phe Glu Asn Phe Leu Ala Thr Lys TrpThr Thr Ala Lys Arg Phe Gly 255 260 265 ctg gaa ggt gct gaa tct ttg attcct ggc atg aag gag atg ttc gat 926 Leu Glu Gly Ala Glu Ser Leu Ile ProGly Met Lys Glu Met Phe Asp 270 275 280 agg tct gca gat ctc ggg gta gagaac ata gtt atc ggt atg ccc cat 974 Arg Ser Ala Asp Leu Gly Val Glu AsnIle Val Ile Gly Met Pro His 285 290 295 agg ggt cga ctt aat gtt ttg ggtaat gtt gtt aga aaa cct cta cgc 1022 Arg Gly Arg Leu Asn Val Leu Gly AsnVal Val Arg Lys Pro Leu Arg 300 305 310 315 caa ata ttc agc gag ttt agcggt ggt act agg cca gta gat gaa gtt 1070 Gln Ile Phe Ser Glu Phe Ser GlyGly Thr Arg Pro Val Asp Glu Val 320 325 330 ggg ctt tac acc gga aca ggtgat gtg aaa tac cac ttg ggt aca tct 1118 Gly Leu Tyr Thr Gly Thr Gly AspVal Lys Tyr His Leu Gly Thr Ser 335 340 345 tat gat cgt cca act aga ggaggc aaa cat ctc cac ttg tct ttg gta 1166 Tyr Asp Arg Pro Thr Arg Gly GlyLys His Leu His Leu Ser Leu Val 350 355 360 gca aat ccc agt cac ttg gaagca gta gat cct gtt gtg ata ggt aaa 1214 Ala Asn Pro Ser His Leu Glu AlaVal Asp Pro Val Val Ile Gly Lys 365 370 375 acc aga gcg aaa caa tat tacacg aaa gac gag aac aga aca aag aac 1262 Thr Arg Ala Lys Gln Tyr Tyr ThrLys Asp Glu Asn Arg Thr Lys Asn 380 385 390 395 atg ggt att ttg atc catggg gat ggt agc ttt gcc gga caa gga gtg 1310 Met Gly Ile Leu Ile His GlyAsp Gly Ser Phe Ala Gly Gln Gly Val 400 405 410 gtg tat gaa act ctc catctt agt gca ctt cct aac tac tgt acc ggt 1358 Val Tyr Glu Thr Leu His LeuSer Ala Leu Pro Asn Tyr Cys Thr Gly 415 420 425 gga aca gtg cac att gtggtg aat aat caa gtg gct ttc aca acc gat 1406 Gly Thr Val His Ile Val ValAsn Asn Gln Val Ala Phe Thr Thr Asp 430 435 440 ccc agg gaa gga agg tcttca cag tat tgc act gat gtt gca aag gct 1454 Pro Arg Glu Gly Arg Ser SerGln Tyr Cys Thr Asp Val Ala Lys Ala 445 450 455 ttg agc gcc cca att ttccat gtc aat gca gat gac att gaa gca gta 1502 Leu Ser Ala Pro Ile Phe HisVal Asn Ala Asp Asp Ile Glu Ala Val 460 465 470 475 gtg cat gct tgt gagctt gct gct gag tgg cgc cag acg ttc cat tct 1550 Val His Ala Cys Glu LeuAla Ala Glu Trp Arg Gln Thr Phe His Ser 480 485 490 gat gtt gtt gtt gattta gta tgc tac cgt cgc ttt ggg cat aac gag 1598 Asp Val Val Val Asp LeuVal Cys Tyr Arg Arg Phe Gly His Asn Glu 495 500 505 ata gac gaa ccg tcattc aca caa cca aaa atg tac aag gtg ata cgc 1646 Ile Asp Glu Pro Ser PheThr Gln Pro Lys Met Tyr Lys Val Ile Arg 510 515 520 agt cat ccc tcg tcactt caa atc tac cag gag aag ctc ttg caa tct 1694 Ser His Pro Ser Ser LeuGln Ile Tyr Gln Glu Lys Leu Leu Gln Ser 525 530 535 gga cag gta acc caagaa gat att gat aag att caa aag aaa gta agc 1742 Gly Gln Val Thr Gln GluAsp Ile Asp Lys Ile Gln Lys Lys Val Ser 540 545 550 555 tct atc ctc aatgaa gaa tat gag gca agt aaa gat tat att cca caa 1790 Ser Ile Leu Asn GluGlu Tyr Glu Ala Ser Lys Asp Tyr Ile Pro Gln 560 565 570 aaa cgt gac tggctg gca agt cac tgg act gga ttc aag tct ccg gag 1838 Lys Arg Asp Trp LeuAla Ser His Trp Thr Gly Phe Lys Ser Pro Glu 575 580 585 cag att tct aggatt cga aac acc gga gtg aag cca gag att ttg aag 1886 Gln Ile Ser Arg IleArg Asn Thr Gly Val Lys Pro Glu Ile Leu Lys 590 595 600 aat gtg gga aaggca atc tca acc ttc cct gag aac ttt aag cca cac 1934 Asn Val Gly Lys AlaIle Ser Thr Phe Pro Glu Asn Phe Lys Pro His 605 610 615 aga gga gtt aaaaga gtt tat gaa caa cgt gct caa atg att gaa tcg 1982 Arg Gly Val Lys ArgVal Tyr Glu Gln Arg Ala Gln Met Ile Glu Ser 620 625 630 635 gga gaa ggcatt gac tgg gga ctt gga gaa gca ctt gct ttt gct aca 2030 Gly Glu Gly IleAsp Trp Gly Leu Gly Glu Ala Leu Ala Phe Ala Thr 640 645 650 ctg gtt gtggaa ggg aac cat gtt cgg cta agt ggt caa gat gtt gaa 2078 Leu Val Val GluGly Asn His Val Arg Leu Ser Gly Gln Asp Val Glu 655 660 665 aga gga actttc agt cat aga cac tca gtg ctt cat gat caa gaa acc 2126 Arg Gly Thr PheSer His Arg His Ser Val Leu His Asp Gln Glu Thr 670 675 680 ggg gag gaatat tgt ccc ctc gat cac cta atc aaa aac caa gac cct 2174 Gly Glu Glu TyrCys Pro Leu Asp His Leu Ile Lys Asn Gln Asp Pro 685 690 695 gaa atg ttcact gtc agc aac agc tcc ctt tca gaa ttt ggt gtt ctc 2222 Glu Met Phe ThrVal Ser Asn Ser Ser Leu Ser Glu Phe Gly Val Leu 700 705 710 715 ggt ttcgaa ctg ggt tat tcg atg gaa aat ccc aat tct ctg gtg ata 2270 Gly Phe GluLeu Gly Tyr Ser Met Glu Asn Pro Asn Ser Leu Val Ile 720 725 730 tgg gaagct cag ttt gga gac ttt gct aat ggc gca caa gtt atg ttt 2318 Trp Glu AlaGln Phe Gly Asp Phe Ala Asn Gly Ala Gln Val Met Phe 735 740 745 gat cagttc ata agc agt ggg gaa gcc aaa tgg ctc cgt caa act ggt 2366 Asp Gln PheIle Ser Ser Gly Glu Ala Lys Trp Leu Arg Gln Thr Gly 750 755 760 cta gtagtt tta ctt cct cat gga tat gat ggt cag ggt cct gaa cat 2414 Leu Val ValLeu Leu Pro His Gly Tyr Asp Gly Gln Gly Pro Glu His 765 770 775 tcc agtgga aga ttg gaa cgt ttc ctt cag atg agt gat gac aat cct 2462 Ser Ser GlyArg Leu Glu Arg Phe Leu Gln Met Ser Asp Asp Asn Pro 780 785 790 795 tacgtt atc cct gag atg gac cca act ctt cga aag cag att caa gaa 2510 Tyr ValIle Pro Glu Met Asp Pro Thr Leu Arg Lys Gln Ile Gln Glu 800 805 810 tgtaat tgg caa gtt gtt aat gtt act aca cct gcc aac tat ttc cat 2558 Cys AsnTrp Gln Val Val Asn Val Thr Thr Pro Ala Asn Tyr Phe His 815 820 825 gttctg cgt cgg cag ata cac agg gac ttt cgc aag cct ctt ata gtg 2606 Val LeuArg Arg Gln Ile His Arg Asp Phe Arg Lys Pro Leu Ile Val 830 835 840 atggcc ccc aaa aac ttg ctt cgt cac aaa cag tgt gta tct aat ctc 2654 Met AlaPro Lys Asn Leu Leu Arg His Lys Gln Cys Val Ser Asn Leu 845 850 855 tcggaa ttc gat gat gtt aaa gga cat cct gga ttt gac aag caa gga 2702 Ser GluPhe Asp Asp Val Lys Gly His Pro Gly Phe Asp Lys Gln Gly 860 865 870 875act cga ttt aaa cgg ttg atc aaa gat caa agt ggc cac tct gat ctt 2750 ThrArg Phe Lys Arg Leu Ile Lys Asp Gln Ser Gly His Ser Asp Leu 880 885 890gaa gaa ggt atc aga cgt cta gtc ctc tgc tct ggg aag gtc tac tat 2798 GluGlu Gly Ile Arg Arg Leu Val Leu Cys Ser Gly Lys Val Tyr Tyr 895 900 905gag ctt gac gaa gag cga aag aag tct gaa aca aag gat gta gcc att 2846 GluLeu Asp Glu Glu Arg Lys Lys Ser Glu Thr Lys Asp Val Ala Ile 910 915 920tgc aga gta gag cag ctt tgc cca ttt cca tat gat ctc atc caa aga 2894 CysArg Val Glu Gln Leu Cys Pro Phe Pro Tyr Asp Leu Ile Gln Arg 925 930 935gaa cta aag cga tat cca aat gca gag atc gtg tgg tgt caa gaa gag 2942 GluLeu Lys Arg Tyr Pro Asn Ala Glu Ile Val Trp Cys Gln Glu Glu 940 945 950955 ccg atg aac atg gga gga tac caa tac ata gcc cta agg ctt tgc acc 2990Pro Met Asn Met Gly Gly Tyr Gln Tyr Ile Ala Leu Arg Leu Cys Thr 960 965970 gcg atg aaa gca ctg caa aga gga aac ttc aac gac atc aaa tac gtt 3038Ala Met Lys Ala Leu Gln Arg Gly Asn Phe Asn Asp Ile Lys Tyr Val 975 980985 ggt cgt ctt ccc tca gct gct aca gcc aca gga ttt tac cag ctt cat 3086Gly Arg Leu Pro Ser Ala Ala Thr Ala Thr Gly Phe Tyr Gln Leu His 990 9951000 gtt aag gag cag act gat ctt gtg aag aaa gct ctt caa cct gac 3131Val Lys Glu Gln Thr Asp Leu Val Lys Lys Ala Leu Gln Pro Asp 1005 10101015 ccc atc acc ccc gtc atc cct taaaaaaaca cagcttgaga ggcttgagcc 3182Pro Ile Thr Pro Val Ile Pro 1020 1025 tgtataaaaa agacacaaca caaaaataaaagattcatga gagaatcttt ggttaccaaa 3242 gagtgtcact ggaaaataaa cagatgtttgctagacttac aaatttaagt ttattcgatt 3302 tgtttggttt gttataggat ttaatcgagataaaaggaaa aaagatttaa accgtttggt 3362 ttagtatgat aattcattaa tttggttcaactaaaaaaaa aaaaaaaaaa 3412 7 1025 PRT Arabidopsis thaliana 7 Met Val TrpPhe Arg Ile Gly Ser Ser Val Ala Lys Leu Ala Ile Arg 1 5 10 15 Arg ThrLeu Ser Gln Ser Arg Cys Gly Ser Tyr Ala Thr Arg Thr Arg 20 25 30 Val LeuPro Cys Gln Thr Arg Cys Phe His Ser Thr Ile Leu Lys Ser 35 40 45 Lys AlaGlu Ser Ala Ala Pro Val Pro Arg Pro Val Pro Leu Ser Lys 50 55 60 Leu ThrAsp Ser Phe Leu Asp Gly Thr Ser Ser Val Tyr Leu Glu Glu 65 70 75 80 LeuGln Arg Ala Trp Glu Ala Asp Pro Asn Ser Val Asp Glu Ser Trp 85 90 95 AspAsn Phe Phe Arg Asn Phe Val Gly Gln Ala Ser Thr Ser Pro Gly 100 105 110Ile Ser Gly Gln Thr Ile Gln Glu Ser Met Arg Leu Leu Leu Leu Val 115 120125 Arg Ala Tyr Gln Val Asn Gly His Met Lys Ala Lys Leu Asp Pro Leu 130135 140 Gly Leu Glu Lys Arg Glu Ile Pro Glu Asp Leu Thr Pro Gly Leu Tyr145 150 155 160 Gly Phe Thr Glu Ala Asp Leu Asp Arg Glu Phe Phe Leu GlyVal Trp 165 170 175 Arg Met Ser Gly Phe Leu Ser Glu Asn Arg Pro Val GlnThr Leu Arg 180 185 190 Ser Ile Leu Ser Arg Leu Glu Gln Ala Tyr Cys GlyThr Ile Gly Tyr 195 200 205 Glu Tyr Met His Ile Ala Asp Arg Asp Lys CysAsn Trp Leu Arg Asp 210 215 220 Lys Ile Glu Thr Pro Thr Pro Arg Gln TyrAsn Ser Glu Arg Arg Met 225 230 235 240 Val Ile Tyr Asp Arg Leu Thr TrpSer Thr Gln Phe Glu Asn Phe Leu 245 250 255 Ala Thr Lys Trp Thr Thr AlaLys Arg Phe Gly Leu Glu Gly Ala Glu 260 265 270 Ser Leu Ile Pro Gly MetLys Glu Met Phe Asp Arg Ser Ala Asp Leu 275 280 285 Gly Val Glu Asn IleVal Ile Gly Met Pro His Arg Gly Arg Leu Asn 290 295 300 Val Leu Gly AsnVal Val Arg Lys Pro Leu Arg Gln Ile Phe Ser Glu 305 310 315 320 Phe SerGly Gly Thr Arg Pro Val Asp Glu Val Gly Leu Tyr Thr Gly 325 330 335 ThrGly Asp Val Lys Tyr His Leu Gly Thr Ser Tyr Asp Arg Pro Thr 340 345 350Arg Gly Gly Lys His Leu His Leu Ser Leu Val Ala Asn Pro Ser His 355 360365 Leu Glu Ala Val Asp Pro Val Val Ile Gly Lys Thr Arg Ala Lys Gln 370375 380 Tyr Tyr Thr Lys Asp Glu Asn Arg Thr Lys Asn Met Gly Ile Leu Ile385 390 395 400 His Gly Asp Gly Ser Phe Ala Gly Gln Gly Val Val Tyr GluThr Leu 405 410 415 His Leu Ser Ala Leu Pro Asn Tyr Cys Thr Gly Gly ThrVal His Ile 420 425 430 Val Val Asn Asn Gln Val Ala Phe Thr Thr Asp ProArg Glu Gly Arg 435 440 445 Ser Ser Gln Tyr Cys Thr Asp Val Ala Lys AlaLeu Ser Ala Pro Ile 450 455 460 Phe His Val Asn Ala Asp Asp Ile Glu AlaVal Val His Ala Cys Glu 465 470 475 480 Leu Ala Ala Glu Trp Arg Gln ThrPhe His Ser Asp Val Val Val Asp 485 490 495 Leu Val Cys Tyr Arg Arg PheGly His Asn Glu Ile Asp Glu Pro Ser 500 505 510 Phe Thr Gln Pro Lys MetTyr Lys Val Ile Arg Ser His Pro Ser Ser 515 520 525 Leu Gln Ile Tyr GlnGlu Lys Leu Leu Gln Ser Gly Gln Val Thr Gln 530 535 540 Glu Asp Ile AspLys Ile Gln Lys Lys Val Ser Ser Ile Leu Asn Glu 545 550 555 560 Glu TyrGlu Ala Ser Lys Asp Tyr Ile Pro Gln Lys Arg Asp Trp Leu 565 570 575 AlaSer His Trp Thr Gly Phe Lys Ser Pro Glu Gln Ile Ser Arg Ile 580 585 590Arg Asn Thr Gly Val Lys Pro Glu Ile Leu Lys Asn Val Gly Lys Ala 595 600605 Ile Ser Thr Phe Pro Glu Asn Phe Lys Pro His Arg Gly Val Lys Arg 610615 620 Val Tyr Glu Gln Arg Ala Gln Met Ile Glu Ser Gly Glu Gly Ile Asp625 630 635 640 Trp Gly Leu Gly Glu Ala Leu Ala Phe Ala Thr Leu Val ValGlu Gly 645 650 655 Asn His Val Arg Leu Ser Gly Gln Asp Val Glu Arg GlyThr Phe Ser 660 665 670 His Arg His Ser Val Leu His Asp Gln Glu Thr GlyGlu Glu Tyr Cys 675 680 685 Pro Leu Asp His Leu Ile Lys Asn Gln Asp ProGlu Met Phe Thr Val 690 695 700 Ser Asn Ser Ser Leu Ser Glu Phe Gly ValLeu Gly Phe Glu Leu Gly 705 710 715 720 Tyr Ser Met Glu Asn Pro Asn SerLeu Val Ile Trp Glu Ala Gln Phe 725 730 735 Gly Asp Phe Ala Asn Gly AlaGln Val Met Phe Asp Gln Phe Ile Ser 740 745 750 Ser Gly Glu Ala Lys TrpLeu Arg Gln Thr Gly Leu Val Val Leu Leu 755 760 765 Pro His Gly Tyr AspGly Gln Gly Pro Glu His Ser Ser Gly Arg Leu 770 775 780 Glu Arg Phe LeuGln Met Ser Asp Asp Asn Pro Tyr Val Ile Pro Glu 785 790 795 800 Met AspPro Thr Leu Arg Lys Gln Ile Gln Glu Cys Asn Trp Gln Val 805 810 815 ValAsn Val Thr Thr Pro Ala Asn Tyr Phe His Val Leu Arg Arg Gln 820 825 830Ile His Arg Asp Phe Arg Lys Pro Leu Ile Val Met Ala Pro Lys Asn 835 840845 Leu Leu Arg His Lys Gln Cys Val Ser Asn Leu Ser Glu Phe Asp Asp 850855 860 Val Lys Gly His Pro Gly Phe Asp Lys Gln Gly Thr Arg Phe Lys Arg865 870 875 880 Leu Ile Lys Asp Gln Ser Gly His Ser Asp Leu Glu Glu GlyIle Arg 885 890 895 Arg Leu Val Leu Cys Ser Gly Lys Val Tyr Tyr Glu LeuAsp Glu Glu 900 905 910 Arg Lys Lys Ser Glu Thr Lys Asp Val Ala Ile CysArg Val Glu Gln 915 920 925 Leu Cys Pro Phe Pro Tyr Asp Leu Ile Gln ArgGlu Leu Lys Arg Tyr 930 935 940 Pro Asn Ala Glu Ile Val Trp Cys Gln GluGlu Pro Met Asn Met Gly 945 950 955 960 Gly Tyr Gln Tyr Ile Ala Leu ArgLeu Cys Thr Ala Met Lys Ala Leu 965 970 975 Gln Arg Gly Asn Phe Asn AspIle Lys Tyr Val Gly Arg Leu Pro Ser 980 985 990 Ala Ala Thr Ala Thr GlyPhe Tyr Gln Leu His Val Lys Glu Gln Thr 995 1000 1005 Asp Leu Val LysLys Ala Leu Gln Pro Asp Pro Ile Thr Pro Val 1010 1015 1020 Ile Pro 10258 20 DNA Artificial Sequence Synthetic DNA 8 atctcattca gcgtgagctc 20 929 DNA Artificial Sequence Synthetic DNA 9 cgggtcgaca gcaataacaaaactgtata 29 10 25 DNA Artificial Sequence Synthetic DNA 10 cgggtacccaagtgtagaac gacga 25 11 28 DNA Artificial Sequence Synthetic DNA 11cgtctagatg gtcggttctc agacatga 28

What is claimed is:
 1. A method for producing a transformed plant whosefree glutamic acid content is increased as compared with a naturallyoccurring plant of the same kind cultivated under the same conditions,comprising the steps of transforming a plant with a nucleic acidconstruct for controlling the expression of 2-oxoglutarate dehydrogenase(OGDH), and then screening the transformed plant based on the phenotypeconferred to the plant by a marker gene present on the nucleic acidconstruct to thus select the transformed plant having an increased freeglutamic acid content.
 2. The method of claim 1, wherein the nucleicacid construct for controlling the expression of 2-oxoglutaratedehydrogenase (OGDH) is a nucleic acid construct comprising an antisenseRNA against the full length of the cDNA sequence of a gene encoding OGDHor a partial sequence thereof and a regulator sequence, which canexpress, in plant's cells, the antisense RNA sequence.
 3. The method ofclaim 1, wherein the nucleic acid construct controls the expression ofOGDH E1 subunit.
 4. The method of claim 1, wherein the nucleic acidconstruct contains the sequence of nucleotide nos.1-3379 of SEQ ID:4 orits complementary sequence.
 5. The method of claim 2, wherein theantisense RNA is an antisense RNA against a coding region in the cDNA ofthe gene coding for OGDH.
 6. The method of claim 2, wherein theantisense RNA is an antisense RNA against OGDH E1 subunit mRNA.
 7. Aplant produced by the method of claim
 1. 8. A plant produced by themethod of claim
 2. 9. A plant produced by the method of claim
 3. 10. Aplant produced by the method of claim
 4. 11. A progeny plant of thetransformed plant of claim 7 and whose free glutamic acid content isimproved as compared with that observed for a naturally occurring plantof the same kind cultivated under the same conditions.
 12. A progenyplant of the transformed plant of claim 8 and whose free glutamic acidcontent is improved as compared with that observed for a naturallyoccurring plant of the same kind cultivated under the same conditions.13. The plant of claim 7, wherein the plant is one belonging toCruciferae.
 14. The plant of claim 8, wherein the plant is one belongingto Cruciferae.
 15. A seed of the plant of claim 1, wherein a nucleicacid construct for controlling the expression of 2-oxoglutaratedehydrogenase (OGDH) is operably incorporated into the genome thereof.16. A seed of the plant of claim 2, wherein a nucleic acid construct forcontrolling the expression of 2-oxoglutarate dehydrogenase (OGDH) isoperably incorporated into the genome thereof.